Skip to main content
NCBI home page
Philosophical Transactions of the Royal Society B: Biological Sciences logoLink to Philosophical Transactions of the Royal Society B: Biological Sciences
. 2019 Jan 14;374(1767):20180314. doi: 10.1098/rstb.2018.0314

Armet, an aphid effector protein, induces pathogen resistance in plants by promoting the accumulation of salicylic acid

Na Cui 1,3,†, Hong Lu 1,†, Tianzuo Wang 2, Wenhao Zhang 2,3, Le Kang 1,3,✉, Feng Cui 1,3,✉
PMCID: PMC6367143  PMID: 30967016

Abstract

Effector proteins present in aphid saliva are thought to modulate aphid–plant interactions. Armet, an effector protein, is found in the phloem sap of pea-aphid-infested plants and is indispensable for the survival of aphids on plants. However, its function in plants has not been investigated. Here, we explored the functions of Armet after delivery into plants. Examination of the transcriptomes of Nicotiana benthamiana and Medicago truncatula following transgenic expression of Armet or infiltration of the protein showed that Armet activated pathways associated with plant–pathogen interactions, mitogen-activated protein kinase and salicylic acid (SA). Armet induced a fourfold increase in SA accumulation by regulating the expression of SAMT and SABP2, two genes associated with SA metabolism, in Armet-infiltrated tobacco. The increase in SA enhanced the plants' resistance to bacterial pathogen Pseudomonas syringae but had no detectable adverse effects on aphid survival or reproduction. Similar molecular responses and a chlorosis phenotype were induced in tobacco by Armet from two aphid species but not by locust Armet, suggesting that the effector function of Armet may be specific for aphids. The results suggest that Armet causes plants to make a pathogen-resistance decision and reflect a novel tripartite insect–plant–pathogen interaction.

This article is part of the theme issue ‘Biotic signalling sheds light on smart pest management’.

Keywords: insect effector, Armet, salicylic acid, pathogen resistance, SAMT, SABP2

1. Introduction

As a group of piercing/sucking insects, aphids probe only mesophyll cells and ingest the cell contents through their stylets. During this process, aphids secrete saliva to enable them to feed stably and efficiently [1]. Aphid saliva is believed to contain effector proteins that potentially have similar functions in plants as their counterparts in pathogenic bacteria, fungi, oomycetes and nematodes [2]. The immune reactions of plants to aphid feeding are similar to their reactions to fungal or bacterial pathogen infection in terms of the plant–pathogen interaction pathways involved [3,4]. Although many proteins have been identified in the saliva of various kinds of aphids [5–7], the functions of most of them as effectors in aphid host plants remain elusive.

Hormone signalling plays a key role in plant immunity. Salicylic acid (SA) and jasmonic acid (JA) are regarded as major defence hormones in plants [8]. JA and jasmonates are ubiquitous signals for tissue injury and for the subsequent activation of plant defense responses to many herbivorous insects. Constitutive activation of JA signalling in Arabidopsis thaliana enhanced plant resistance to Myzus persicae [9], and blocking JA signalling promoted population growth in M. persicae and Brevicoryne brassicae [10]. The role of SA in resistance to piercing/sucking insects is controversial. Intercellular application of SA stimulated the defence responses of wheat against Russian wheat aphid (Diuraphis noxia) [11]. However, overexpression of SA-related genes and mutation of these genes showed that SA signalling was not critical for controlling M. persicae infestation of A. thaliana [3]. SA accumulation, activation of SA-responsive gene expression, and inhibition of JA-responsive gene expression have frequently been observed in plants such as wheat, barley, tomato and A. thaliana after infestation with various species of aphids [12,13]. Nonetheless, the identity of the effector(s) in aphid saliva that stimulates plant SA signalling remains unknown.

In our previous work, we reported that Armet protein of the pea aphid (Acyrthosiphon pisum) (ApArmet) is secreted into the phloem sap of fava beans together with the watery saliva of the aphid during the feeding process. As an effector, ApArmet induced the expression of genes involved in the plant–pathogen interaction pathway after inoculation into Nicotiana benthamiana leaves [14]. However, the molecular mechanisms through which Armet acts as an effector during the regulation of aphid–plant interactions are unknown. In the present study, we investigated the immune responses of two plant species, N. benthamiana and Medicago truncatula, to Armet through extracellular application and intracellular expression. In addition to activating the plant–pathogen interaction and mitogen-activated protein kinases (MAPK) signalling pathways, Armet was found to increase SA accumulation and activate SA-responsive gene expression. The SA accumulation induced by Armet conferred resistance to bacterial pathogens but not to aphids.

2. Material and methods

(a). Plants and aphids

Medicago truncatula R108 and A17 and N. benthamiana were used in the study. Germinated seeds of N. benthamiana and M. truncatula were transplanted to 8 cm × 8 cm or 12-cm-diameter plastic pots filled with a mixture of nutrient soil and vermiculite (1 : 1, v/v) and cultured at 21°C with a long-day photoperiod (16 L : 8D) at 70% relative humidity. Populations of M. persicae and A. pisum were raised on A. thaliana and M. truncatula A17, respectively, in a growth chamber with a 16-h light photoperiod at 23°C.

(b). RNA extraction and real-time quantitative PCR

Total RNA was isolated from N. benthamiana and M. truncatula leaves and from M. persicae and Locusta migratoria, and then was reverse-transcribed to cDNA. Real-time quantitative PCR (qPCR) was used to quantify the transcript levels of various genes in N. benthamiana and M. truncatula. qPCR was performed in a Light Cycler 480 II instrument (Roche, Basel, Switzerland). Differences were analysed using one-way ANOVA for multiple comparisons or the t-test for pairwise comparisons in SPSS 17.0. Detailed procedures can be found in electronic supplementary material, Materials and Methods.

(c). Generation of transgenic N. benthamiana and M. truncatula expressing ApArmet

To generate transgenic N. benthamiana for ApArmet transient expression, ApArmet (XM_001949506 in GenBank) lacking the sequence encoding the signal peptide was cloned in the pENTR/D-TOPO vector using the pENTR/D-TOPO Cloning Kit (Invitrogen, Carlsbad, CA, USA) and the primer pair ApArmet-F/ApArmer-R (electronic supplementary material, table S1); it was then recombined with the destination vector pFAST-G02 using LR Clonase Enzyme Mix (Invitrogen). The ApArmet transient expression assay was performed by infiltrating four-week-old N. benthamiana leaves with Agrobacterium tumefaciens strain GV3101 (OD600 nm = 1) carrying the recombinant destination vector. Leaves were collected at 66 h post-inoculation (hpi), and the transcript level of ApArmet was measured by qPCR. The pFAST-G02 empty vector was transformed as a negative control.

For the constitutive expression of ApArmet in M. truncatula R108, ApArmet lacking the sequence encoding the signal peptide was cloned in the pMDC32 Gateway vector with a 2 × 35S promoter using the primers ApArmet-KpnI-F/ApArmet-SpeI-R to generate the recombinant destination vector (electronic supplementary material, table S1). Agrobacterium transformation and the regeneration of M. truncatula R108 via somatic embryogenesis have been described previously [15]. For the detection of ApArmet in F1 plants, the ApArmet transgene was amplified by PCR using the primers ApArmet-q-F/ApArmet-q-R and genomic DNA that had been extracted from the leaves using the cetyltrimethylammonium ammonium bromide (CTAB) method. The expression level of ApArmet in positive transgenic F1 plants was measured by qPCR. The F1 plants with high ApArmet expression levels were self-fertilized. After determining the genotype separation ratio and the ApArmet expression level in the F3 generation, one line of the F2 generation of plants was selected for transcriptome sequencing. Native M. truncatula R108 was used as a negative control.

(d). Protein expression, purification and infiltration of leaves

The cloning, expression and purification of ApArmet and the Armets from M. persicae (MpArmet) and L. migratoria (LmArmet) are described in electronic supplementary material, Materials and Methods. After dilution to a final concentration of 25 ng µl−1 or 50 ng µl−1 in buffer (20 mM Tris–HCl, 120 mM NaCl, pH 8.0), 100 µl purified ApArmet, MpArmet or LmArmet was infiltrated into four-week-old N. benthamiana leaves using a sterile 1-ml syringe and a 0.4 × 13 RWLB needle (Shanghai Misawa Medical Industry, Shanghai, China). The plants were photographed daily for 14 days. After concentration to 100 ng µl−1 using a Millipore ultrafiltration device, 5 µl of purified ApArmet protein was infiltrated into eight-week-old M. truncatula R108 leaves by microinjection through a glass needle at slow speed using a Nanoliter 2000 device (World Precision Instruments, Sarasota, FL, USA). An equal volume of purified product obtained from Escherichia coli transfected with the pET28a empty vector was infiltrated as a negative control. The leaves of N. benthamiana (25 ng µl−1 ApArmet), M. truncatula with ApArmet infiltration and the control group were collected at 60 hpi for transcriptome sequencing and qPCR.

(e). Transcriptomic sequencing and analysis

Total RNAs were sent to the BGI Company (Shenzhen, China) for RNA-seq analysis using the single-end digital gene expression sequencing strategy. At least 12 million clean reads were obtained for each sample. Reads of each sample were deposited in the Short Read Archive of the National Center for Biotechnology Information (NCBI) under the accession number SRP149658. Differentially expressed genes were analysed with a fold change threshold ≥ 2 and divergence probability ≥ 0.8. Detailed procedures can be found in electronic supplementary material, Materials and Methods. (SAMT, salicylate carboxymethyltransferase; SABP2, salicylic acid-binding protein 2; SAGT, SA glucosyltransferase.)

(f). Knockdown and overexpression of salicylate carboxymethyltransferase and salicylic acid binding protein 2 in tobacco leaves

The open reading frames (ORFs) of SAMT and SABP2 were amplified from the cDNA of N. benthamiana using the primer pairs SAMT-F/SAMT-R and SABP2-F/SABP2-R, respectively, and inserted into the Gateway destination vector pEarleyGate100 using LR Clonase (Invitrogen) for overexpression (oeSAMT, oeSABP2; electronic supplementary material, table S1). An artificial microRNA corresponding to SABP2 (amiRNA-SABP2) was designed and synthesized through several rounds of PCR using the primers SABP2-I miR-s, SABP2-II miR-a, SABP2-III miR*s, SABP2-IV miR*a, SABP2-A and SABP2-B (electronic supplementary material, table S1) based on the protocol described at http://wmd3.weigelworld.org and inserted into the pENTR/D-TOPO vector (Invitrogen). After sequence confirmation, amiRNA-SABP2 was recombined into pEarleyGate100 using LR Clonase (Invitrogen) for SABP2-knockdown (miSABP2). The recombinant plasmids containing oeSAMT, oeSABP2 and miSABP2 were introduced into N. benthamiana leaves using A. tumefaciens strain GV3101 with the empty pEarleyGate100 vector as a negative control. Eighteen hours after introduction, the leaves were infiltrated with 2.5 µg purified ApArmet; the leaves were harvested 48 h later for measurement of gene expression and SA content. An equal volume of purified product obtained from the pET28a empty vector was infiltrated as a control. Six biological replicates with four leaves in each replicate were prepared.

(g). Measurement of salicylic and jasmonic acids

The SA and JA content of ApArmet protein-infiltrated N. benthamiana leaves at 60 hpi was measured after grinding the leaves in liquid nitrogen. The SA content of ApArmet, oeSAMT, oeSABP2 and miSABP2 transgenic N. benthamiana leaves was measured at 66 hpi. SA and JA assays were conducted at the National Centre for Plant Gene Research (Beijing, China) using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with single solid-phase extraction (SPE), purification and isotope dilution as previously described [16]. Three replicates were prepared for each group. Differences between the groups were statistically analysed using the independent-sample t-test in SPSS v. 17.0.

(h). Survival and reproduction of aphids on ApArmet protein-infiltrated plants

After 24 h of infiltration of N. benthamiana and M. truncatula leaves with purified ApArmet, 10–15 first-instar nymphs of M. persicae and 15–18 third-instar nymphs of A. pisum were raised on the N. benthamiana and M. truncatula leaves, respectively, for 13 days. Leaves infiltrated with the purified product obtained from the pET28a empty vector were used as a negative control. Six biological replicates were prepared for each group. The survival rate and the number of offspring were recorded daily. The survival curves of the ApArmet protein-infiltrated and control groups were calculated using Kaplan–Meier method and statistical differences were analysed with the log-rank test in SPSS v. 17.0. The reproductive rate was expressed as the average number of offspring per aphid during the first 4 days of the oviposition period; differences were evaluated using the t-test in SPSS v. 17.0.

(i). Pseudomonas syringae infection

Pseudomonas syringae pv. tabaci was cultured in King's B medium at 28°C for 24 h. After centrifugation at 4000 r.p.m. for 10 min, the bacterial cells were resuspended in 10 mM MgSO4 to a final density of 102 colony-forming units (cfu)/ml. One day after ApArmet infiltration, one tobacco leaf was infiltrated with 100 µl of bacterial suspension at two sites. At 6 and 9 d post-infiltration (dpi) with bacteria, two 1-cm-diameter leaf discs covering the infiltration sites were excised, ground and suspended in 1 ml of 10 mM MgCl2, and 100 µl of the resulting suspension was plated on King's B agar plates and cultured for 24 h at 28°C before the measurement of bacterial cfu. Leaves infiltrated with purified product obtained from the pET28a empty vector were used as a control. Six biological replicates were prepared; the differences between the ApArmet-infiltration and control groups were evaluated using the t-test in SPSS v. 17.0.

(j). Phylogenetic analysis

One hundred and thirty-three insect Armet homologous protein sequences were acquired via a BLASTP search of the non-redundant protein sequence database and a TBLASTN search of the expressed sequence tags in NCBI (http://www.ncbi.nlm.nih.gov/). The Armet sequences of Hyalopterus persikonus, Sitobion avenae and Rhopalosiphum padi were obtained from our previous transcriptomes [17,18]. Protein sequences were aligned using ClustalW, and an unrooted phylogenetic tree was constructed using the neighbour-joining method (p-distance and pairwise deletion) in MEGA 6.0. Bootstrap analysis of 1000 replicates was applied to evaluate the confidence level of the tree topology.

3. Results

(a). Plant responses to Armet

To investigate the function of Armet protein in plants, we generated transgenic M. truncatula that constitutively expressed ApArmet and transgenic N. benthamiana that transiently expressed ApArmet. ApArmet was expressed in vitro, purified and leaves of M. truncatula and N. benthamiana were infiltrated with 0.5 µg and 2.5 µg purified protein, respectively (figure 1a). The four groups of plants and their respective negative controls were subjected to transcriptomic analysis. In transgenic M. truncatula and N. benthamiana, 2105 and 1619 genes, respectively, were differentially expressed, whereas in protein-infiltrated M. truncatula and N. benthamiana, 1996 and 3053 genes, respectively, were differentially expressed (figure 1b). The overall variation in gene expression between the transgenic and protein-infiltrated plants was high (figure 1c). Only a small number of genes showed similar variation in expression in the transgenic and protein-infiltrated plants, i.e. there were 99 co-upregulated genes and 118 co-downregulated genes in M. truncatula and 165 co-upregulated genes and 93 co-downregulated genes in N. benthamiana.

Figure 1.

Figure 1.

Open in a new tab

Transcriptomic analyses of ApArmet transgenic and protein-infiltrated Medicago truncatula and Nicotiana benthamiana. (a) Schematic showing the treatment of the two plants. (b) Number of upregulated and downregulated genes in the four groups of plants compared to the control groups. (c) Heat maps of the differentially expressed genes in the four groups of plants compared to the control groups. The heat maps show the fold change values on a log2 scale. Gene expression is shown as upregulated (red), downregulated (green) or no change (black). 1, ApArmet protein infiltration; 2, ApArmet transgenic expression. (d) Number and fold change in the expression of upregulated and downregulated genes in the plant–pathogen interaction, MAPK, and plant hormone signal transduction pathways. Each line represents a gene.

The functional annotations for the differentially expressed genes included three KEGG pathways associated with pathogen or insect resistance in plants, i.e. the plant–pathogen interaction pathway, the MAPK signalling pathway and plant hormone signal transduction. The number and fold change in expression of the differentially expressed genes in these three KEGG pathways were comparable in the four groups of plants (figure 1d). Plant hormone signal transduction is one of the downstream components of the plant–pathogen interaction pathway and the MAPK signalling pathway. In N. benthamiana and M. truncatula, infiltration of ApArmet protein or transgenic expression of ApArmet activated the SA pathway by upregulating the expression of transcription factor TGA, pathogenesis-related protein 1 (PR1) and regulatory protein NPR1, and suppressed the JA pathway by upregulating the expression of JAZ protein, a transcriptional repressor of JA signalling.

(b). Armet increases salicylic acid levels in plants

After observing the effects of ApArmet on the expression of genes associated with the SA and JA pathways, we measured the concentrations of SA and JA in N. benthamiana using UPLC-MS/MS. The SA concentration in ApArmet protein-infiltrated tobacco plants was fourfold that found in control plants (figure 2a), whereas it did not change in the transgenic plants (figure 2e). We quantified the transcript levels of two SA-responsive genes, PR1 and beta-1,3-glucanase 2 (BGL2) [19] using qPCR. The transcript levels of PR1 and BGL2 in protein-infiltrated tobacco plants increased sixfold and 13-fold, respectively (figure 2b,c), whereas PR1 expression was doubled in the transgenic tobacco (figure 2f,g). Although the amount of SA in the ApArmet protein-infiltrated tobacco plants increased, no change in the JA concentration in these plants was observed, probably because the basal level of JA in N. benthamiana is too low to be detected using our measurement platform (electronic supplementary material, figure S1). The transcript level of defensin 1.2 (PDF1.2), a JA-responsive gene [8], was quantified and found to be downregulated significantly in the ApArmet protein-infiltrated tobacco plants and to remain unchanged in the transgenic tobacco plants (figure 2d,h). These results demonstrate that ApArmet stimulates SA signalling.

Figure 2.

Figure 2.

Open in a new tab

Effects of Armet on SA and JA signalling pathways in Nicotiana benthamiana. (a) SA concentration in N. benthamiana after ApArmet protein infiltration. (b–d) Relative transcript levels of the SA downstream genes PR1 and BGL2 and the JA downstream gene PDF1.2 in N. benthamiana after ApArmet protein infiltration measured using real-time quantitative PCR (qPCR). (e) SA concentration in ApArmet transgenic N. benthamiana. (f–h) Relative transcript levels of PR1, BGL2 and PDF1.2 in ApArmet transgenic N. benthamiana measured using qPCR. The transcript level of each gene relative to that of ef1α is shown as the mean ± s.e. Control 1, plant infiltrated with the purified product from E. coli transfected with pET28a empty vector; Control 2, plant transformed with the pFAST-G02 empty vector. *p < 0.05; **p < 0.01; n.s., no significant difference.

(c). Armet regulates the expression of genes associated with salicylic acid metabolism

Why does Armet facilitate the accumulation of SA in plants? To answer this question, we measured the variation in the expression of genes involved in SA metabolism: SAMT, SABP2 and SA glucosyltransferase (SAGT); SA signal transduction: enhanced disease susceptibility 1 (EDS1) and phytoalexin deficient 4 (PAD4); and SA biosynthesis: isochorismate synthase (ICS) and chalcone synthase (CHS) [20] using qPCR in tobacco plants infiltrated with ApArmet protein. Only two metabolism genes, SAMT and SABP2, and one signal transduction gene, EDS1, showed significant changes in expression in the presence of Armet; Armet downregulated the expression of SAMT and upregulated the expression of SABP2 and EDS1 (figure 3a). The expression of two SA biosynthesis genes, ICS and CHS, did not change in the presence of Armet (figure 3a).

Figure 3.

Figure 3.

Open in a new tab

Armet regulates the expression of genes associated with SA metabolism. (a) The relative transcript levels of genes involved in the metabolism (SAMT, SABP2 and SAGT), signal transduction (EDS1 and PAD4) and biosynthesis (ICS and CHS) of the SA pathway in ApArmet protein-infiltrated Nicotiana benthamiana. (b) Relative transcript levels of SAMT and SABP2 and SA concentrations in SAMT-overexpressing (oeSAMT) and SABP2-overexpressing (oeSABP2) transgenic N. benthamiana. (c) The SA concentrations in oeSAMT and SABP2-knockdown (miSABP2) transgenic N. benthamiana and the relative transcript level of SABP2 in miSABP2 transgenic N. benthamiana after ApArmet protein infiltration. The transcript level of each gene relative to that of ef1α is shown as the mean ± s.e. Control 1, plant infiltrated with the purified product from the E. coli transfected with pET28a empty vector; Control 2, plant transformed with the pEarleyGate100 empty vector. *p < 0.05; **p < 0.01; n.s., no significant difference. Different lowercase letters indicate significant differences at the p < 0.05 level.

SAMT is a methyltransferase that converts SA to methyl salicylate, and SABP2 is a methyl esterase that converts methyl salicylate to SA [20]. To verify the roles of SAMT and SABP2 in the Armet-induced accumulation of SA, we transiently overexpressed SAMT (oeSAMT) and SABP2 (oeSABP2), and knocked down SABP2 expression (miSABP2) in tobacco plants. In the oeSAMT and oeSABP2 tobacco, the transcript levels of SAMT and SABP2, respectively, greatly increased (figure 3b). The content of SA was significantly reduced in the oeSAMT plants and significantly enhanced in the oeSABP2 plants (figure 3b), confirming the functions of these two proteins in the metabolism of SA in tobacco. In the miSABP2 tobacco plants, the transcript level of SABP2 was reduced even in the presence of ApArmet (figure 3c). When ApArmet was infiltrated into transgenic oeSAMT or miSABP2 tobacco plants, SA accumulation was significantly lower than that in non-transgenic plants but still higher than that in control plants without ApArmet (figure 3c). These results indicate that the Armet-induced SA accumulation was due to the downregulation of SAMT and the upregulation of SABP2.

(d). Armet enhances plant resistance to bacterial pathogen P. syringae but not to aphids

As a major defence hormone, SA may affect the fitness of insects or pathogens for growth on plants. Does the increased SA accumulation caused by Armet have effects on aphids or pathogens? We tested the performance of M. persicae on ApArmet protein-infiltrated tobacco leaves and that of A. pisum on ApArmet protein-infiltrated M. truncatula leaves. Neither the survival rate nor the reproductive rate of M. persicae and A. pisum changed when the aphids lived on Armet-infiltrated leaves rather than control leaves (electronic supplementary material, figure S2A-2D). At the same time, we measured the resistance of tobacco plants infiltrated with ApArmet to the bacterial pathogen P. syringae pv. tabaci by monitoring bacterial propagation in the leaves. The number of P. syringae pv. tabaci colonies obtained from ApArmet protein-infiltrated tobacco leaves was significantly reduced at 6 and 9 dpi (electronic supplementary material, figure S2E, 2F); it was only half of that obtained from the control group without ApArmet at 6 dpi. These results show that the increased level of SA produced after ApArmet infiltration enhances plant resistance to bacterial pathogens but not to aphids.

(e). The effector functions of Armet are specific for aphid species

Armet is present in animal species ranging from invertebrates to mammals [14]. We compared the sequences of Armet from 133 insect species. Phylogenetic analysis showed that the Armet sequences from seven aphid species clustered together and that these sequences diverged greatly from the homologous sequences of other insect species (electronic supplementary material, figure S3A). The homology of the amino acid sequences of Armet from the seven aphid species was quite high, with identities greater than 95%. On the other hand, aphid Armet was largely differentiated from Armet of other insects, with which it displayed identities ranging from 50 to 60%. Compared to the carboxy-terminal region, the N-terminal portion of Armet was quite conserved within aphids but more divergent from that of other insects (electronic supplementary material, figure S3B).

To determine whether the functions of Armet are specific for aphid species, we cloned Armet sequences from the green peach aphid M. persicae (MpArmet) and the locust Locusta migratoria (LmArmet). After expression in vitro, purified MpArmet, LmArmet and ApArmet were infiltrated into tobacco N. benthamiana leaves. The expression levels of 18 genes in the plant–pathogen interaction pathway [14] and of PR1, BGL2 and PDF1.2 were quantified using qPCR and compared in protein-infiltrated and control tobacco plants. All 18 plant–pathogen interaction genes, as well as PR1 and BGL2, were upregulated, and PDF1.2 was downregulated by ApArmet or MpArmet, whereas only five plant–pathogen interaction genes were upregulated by LmArmet (figure 4a). Furthermore, PR1, BGL2 and PDF1.2 expression did not respond to LmArmet (figure 4a), indicating that, unlike aphid Armet, LmArmet did not influence SA or JA signalling transduction pathway.

Figure 4.

Figure 4.

Open in a new tab

The effector functions of Armet are specific for aphid species. (a) Fold changes in the expression of genes of the plant–pathogen interaction pathway in Nicotiana benthamiana leaves infiltrated with aphid Armet (ApArmet and MpArmet) or locust Armet (LmArmet) and measured using real-time quantitative PCR. Leaves infiltrated with purified product from the pET28a empty vector were used as a control. *p < 0.05; **p < 0.01; n.s., no significant difference. (b) Chlorosis phenotype of N. benthamiana leaves at different days post-infiltration (dpi) of ApArmet, MpArmet or LmArmet protein. Leaves infiltrated with purified product from the pET28a empty vector were used as a control. The arrows indicate chlorosis. (c) Number and fold change in the expression of upregulated and downregulated genes in three photosynthesis-related pathways in the transcriptome of ApArmet protein-infiltrated N. benthamiana. Each line represents a gene.

In addition to measuring the gene expression response to the three Armet proteins, we also explored the phenotypes induced by these proteins in the leaves of tobacco N. benthamiana. A similar chlorosis phenotype of leaves was observed at 9 dpi after infiltration of the leaves with 2.5 µg ApArmet or MpArmet; when the amount of protein applied was increased to 5 µg, the leaves became wilted at 14 dpi (figure 4b). By contrast, no obvious symptoms appeared in leaves infiltrated with 2.5 or 5 µg of LmArmet or in control leaves (figure 4b). The chlorosis phenotype induced by ApArmet or MpArmet may be related to the negative effects of the proteins on the photosynthesis system. Examination of the transcriptome of ApArmet protein-infiltrated N. benthamiana showed that the expression of a large number of genes associated with the photosynthesis-antenna, photosynthesis and carbon fixation pathways (especially the photosynthesis-antenna pathway), was downregulated; 34 genes encoding chlorophyll a/b binding proteins were downregulated (figure 4c).

The results reported here show that the molecular response and the phenotype induced by the two aphid Armet proteins were similar, whereas locust Armet did not induce a strong response in plants. These findings indicate that the functions of Armet may be specific for aphid species.

4. Discussion

The immune responses of plants to aphid infestation are similar to the responses to pathogen infection, especially with respect to plant hormone signalling. The activation of plant SA signalling is the convergent point in the response to aphid infestation and pathogen infection. Prior to this study, the factors through which aphids induce SA signalling were unknown. The effects of SA signalling on aphids and other pathogens may differ. The accumulation of SA is detrimental to plant pathogens, but it is not clear that it is detrimental to aphid infestation. Our work identified the first known effector protein found in aphid saliva, Armet, that is capable of inducing an SA response to aphid feeding. Although Armet-induced SA accumulation was not harmful to aphid fitness, it conferred resistance to infection by the bacterial pathogen P. syringae, indicating that aphids deceive plants and cause them to make a pathogen-resistance decision.

Armet promotes the accumulation of SA, thereby increasing the resistance of plants to bacterial pathogens. JA is thought to be an efficient anti-insect hormone, whereas SA plays a key role in pathogen resistance and confers a less effective defence against insects than JA [21]. In most cases, the production of SA and JA is antagonistic [22]. Aphids use Armet to drive plant responses in the direction of an inefficient SA defence against aphids; this is reflected in the unimpaired survival rates and reproductive rates of M. persicae and A. pisum living on Armet-infiltrated leaves with elevated SA accumulation. Similar tripartite insect–plant–pathogen interactions have been observed. For example, feeding of the silverleaf whitefly Bemisia argentifolii on tomato significantly reduces the incidence of powdery mildew [23]. Infestation of rice by the white-backed planthopper Sogatella furcifera induces resistance to the rice blast fungus Magnaporthe grisea [24]. These resistance reactions are attributed to the accumulation of pathogenesis-related proteins or secondary metabolites induced by insect infestation.

Armet regulates the metabolism of SA via SAMT and SABP2. Armet does not influence the expression of SA synthesis genes; instead, it regulates the expression of SAMT and SABP2 to promote SA accumulation. SAMT belongs to the SABATH family of methytransferases, which contains 24 members in Arabidopsis [25]. One homologous protein, NtSAMT of Nicotiana tabacum, converts SA to MeSA and has a high affinity for SA. Silencing of NtSAMT reduced MeSA levels in primary leaves and blocked systematic acquired resistance to tobacco mosaic virus [26]. The MeSA esterase activity of SABP2 (NtSABP2) has also been demonstrated in N. tabacum [27]. NtSAMT and NtSABP2 are the main enzymes that contribute to systemic acquired resistance in N. tabacum. Arabidopsis and Solanum tuberosum possess 18 and one orthologous SABP2 genes, respectively. Only one SABP2 gene and 12 genes of the SABATH family were identified in the genome of N. benthamiana. In the present study, we cloned one SAMT gene and the unique SABP2 gene of N. benthamiana and verified their functions in the metabolism of SA for the first time. Furthermore, the expression of these two genes was found to be regulated by aphid Armet. How aphid Armet regulates the expression of these two genes is a subject worthy of further research.

Aphid effectors play diverse roles in aphid–plant interactions. Armet and C002 from A. pisum are indispensable for normal feeding behaviour and survival of aphids on plants. Overexpression of C002 in plants enhanced aphid colonization, while infiltration of Armet protein did not affect aphid fitness on plants [28]. Two effectors (Me10 and Me23) of the potato aphid Macrosiphum euphorbiae enhanced aphid fecundity when delivered into N. benthamiana [29], whereas expression of the M. persicae effectors Mp10, Mp42, Mp56, Mp57 and Mp58 in N. benthamiana decreased aphid reproduction [30,31]. It is worth noting that Mp10 induced chlorosis and local cell death in tobacco from 2 dpi onward [32]. This chlorosis response was dependent on the ubiquitin ligase-associated protein SGT1, which is required for the activation of NBS-LRR proteins and plant resistance responses [33]. By contrast, Armet-induced chlorosis in tobacco appeared at 9 dpi, much later than Mp10-mediated chlorosis. We measured the SGT1 transcript level and found that the expression of this gene did not respond to Armet (data not shown). Armet-induced chlorosis may result from downregulation of the expression of chlorophyll a/b binding proteins, which are involved in the function of light-harvesting complexes I and II. The downregulation of chlorophyll a/b binding proteins decreases chlorophyll levels in Arabidopsis [34]. Therefore, the mechanisms underlying the chlorosis phenotype differ for Armet and Mp10.

In conclusion, aphids exploit Armet, a ubiquitous animal protein, as an effector to suppress the effective JA pathway through the modification of the SA pathway to benefit their feeding activity and increase plant resistance to bacterial pathogens. This function is likely specific to aphids. These results reflect an adaptation strategy used by aphids to exploit tripartite insect–plant–pathogen interactions.

Supplementary Material

Supplementary material
rstb20180314supp1.pdf (787.8KB, pdf)

Acknowledgements

We thank Dr. Jian Ye and Dr. Guixian Xia of the Institute of Microbiology, Chinese Academy of Sciences, for providing Pseudomonas syringae pv. tabaci and for guidance on bacterial infection.

Data accessibility

The data cited in this research are available from the Short Read Archive of NCBI accession no. SRP149658.

Authors' contributions

N.C. and H.L. performed the experiments. T.W. and W.Z. generated the transgenic M. truncatula. F.C. and L.K. designed the experiments. N.C., H.L. and F.C. wrote the manuscript.

Competing interests

We have no competing interests.

Funding

This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDB11040200) and by the Natural Science Foundation of China (No. 31772162).

References

  • 1.Will T, Tjallingii WF, Thonnessen A, van Bel AJE. 2007. Molecular sabotage of plant defense by aphid saliva. Proc. Natl Acad. Sci. USA 104, 10 536–10 541. ( 10.1073/pnas.0703535104) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Dangl JL, Horvath DM, Staskawicz BJ. 2013. Pivoting the plant immune system from dissection to deployment. Science 341, 746–751. ( 10.1126/science.1236011) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Louis J, Shah J. 2013. Arabidopsis thaliana-Myzus persicae interaction: shaping the understanding of plant defense against phloem-feeding aphids. Front. Plant Sci. 4, 213 ( 10.3389/fpls.2013.00213) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Rodriguez PA, Bos JIB. 2013. Toward understanding the role of aphid effectors in plant infestation. Mol. Plant Microbe Interact. 26, 25–30. ( 10.1094/Mpmi-05-12-0119-Fi) [DOI] [PubMed] [Google Scholar]
  • 5.Carolan JC, et al. 2011. Predicted effector molecules in the salivary secretome of the pea aphid (Acyrthosiphon pisum): a dual transcriptomic/proteomic approach. J. Proteome Res. 10, 1505–1518. ( 10.1021/Pr100881q) [DOI] [PubMed] [Google Scholar]
  • 6.Cooper WR, Dillwith JW, Puterka GJ. 2011. Comparisons of salivary proteins from five aphid (Hemiptera: Aphididae) species. Environ. Entomol. 40, 151–156. ( 10.1603/EN10153) [DOI] [PubMed] [Google Scholar]
  • 7.Vandermoten S, Harmel N, Mazzucchelli G, De Pauw E, Haubruge E, Francis F.. 2014. Comparative analyses of salivary proteins from three aphid species. Insect Mol. Biol. 23, 67–77. ( 10.1111/imb.12061) [DOI] [PubMed] [Google Scholar]
  • 8.Pieterse CMJ, Van der Does D, Zamioudis C, Leon-Reyes A, Van Wees SCM. 2012. Hormonal modulation of plant immunity. Annu. Rev. Cell Dev. Biol. 28, 489–521. ( 10.1146/annurev-cellbio-092910-154055) [DOI] [PubMed] [Google Scholar]
  • 9.Ellis C, Karafyllidis L, Turner JG. 2002. Constitutive activation of jasmonate signaling in an Arabidopsis mutant correlates with enhanced resistance to Erysiphe cichoracearum, Pseudomonas syringae, and Myzus persicae. Mol. Plant Microbe Interact. 15, 1025–1030. ( 10.1094/Mpmi.2002.15.10.1025) [DOI] [PubMed] [Google Scholar]
  • 10.Mewis I, Tokuhisa JG, Schultz JC, Appel HM, Ulrichs C, Gershenzon J. 2006. Gene expression and glucosinolate accumulation in Arabidopsis thaliana in response to generalist and specialist herbivores of different feeding guilds and the role of defense signaling pathways. Phytochemistry 67, 2450–2462. ( 10.1016/j.phytochem.2006.09.004) [DOI] [PubMed] [Google Scholar]
  • 11.Mohase L, van der Westhuizen AJ.. 2002. Salicylic acid is involved in resistance responses in the Russian wheat aphid-wheat interaction. J. Plant Physiol. 159, 585–590. ( 10.1078/0176-1617-0633) [DOI] [PubMed] [Google Scholar]
  • 12.Chaman ME, Copaja SV, Argandona VH. 2003. Relationships between salicylic acid content, phenylalanine ammonia-lyase (PAL) activity, and resistance of barley to aphid infestation. J. Agric. Food Chem. 51, 2227–2231. ( 10.1021/jf020953b) [DOI] [PubMed] [Google Scholar]
  • 13.Cooper WC, Jia L, Goggin FL. 2004. Acquired and R-gene-mediated resistance against the potato aphid in tomato. J. Chem. Ecol. 30, 2527–2542. ( 10.1007/s10886-004-7948-9) [DOI] [PubMed] [Google Scholar]
  • 14.Wang W, et al. 2015. Armet is an effector protein mediating aphid-plant interactions. FASEB J. 29, 2032–2045. ( 10.1096/fj.14-266023) [DOI] [PubMed] [Google Scholar]
  • 15.Chabaud M, Ratet P, de Sousa Araújo S, Roldão Lopes Amaral Duque AS, Harrison M, Barker DG. 2007. Agrobacterium tumefaciens-mediated transformation and in vitro plant regeneration of M. truncatula. In Medicago truncatula handbook, March 2007 edn, https://www.noble.org/medicago-handbook/2007. [Google Scholar]
  • 16.Fu JH, Chu JF, Sun XH, Wang JD, Yan CY. 2012. Simple, rapid, and simultaneous assay of multiple carboxyl containing phytohormones in wounded tomatoes by UPLC-MS/MS using single SPE purification and isotope dilution. Anal. Sci. 28, 1081–1087. ( 10.2116/analsci.28.1081) [DOI] [PubMed] [Google Scholar]
  • 17.Cui N, Yang PC, Guo K, Kang L, Cui F. 2017. Large-scale gene expression reveals different adaptations of Hyalopterus persikonus to winter and summer host plants. Insect Sci. 24, 431–442. ( 10.1111/1744-7917.12336) [DOI] [PubMed] [Google Scholar]
  • 18.Zhang LP, Lu H, Guo K, Yao SM, Cui F. 2017. Insecticide resistance status and detoxification enzymes of wheat aphids Sitobion avenae and Rhopalosiphum padi. Sci. China Life Sci. 60, 927–930. ( 10.1007/s11427-017-9105-x) [DOI] [PubMed] [Google Scholar]
  • 19.Zarate SI, Kempema LA, Walling LL. 2007. Silverleaf whitefly induces salicylic acid defenses and suppresses effectual jasmonic acid defenses. Plant Physiol. 143, 866–875. ( 10.1104/pp.106.090035) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Vlot AC, Dempsey DA, Klessig DF. 2009. Salicylic acid, a multifaceted hormone to combat disease. Annu. Rev. Phytopathol. 47, 177–206. ( 10.1146/annurev.phyto.050908.135202) [DOI] [PubMed] [Google Scholar]
  • 21.Giordanengo P, Brunissen L, Rusterucci C, Vincent C, van Bel A, Dinant S, Girousse C, Faucher M, Bonnemain JL.. 2010. Compatible plant-aphid interactions: how aphids manipulate plant responses. C. R. Biol. 333, 516–523. ( 10.1016/j.crvi.2010.03.007) [DOI] [PubMed] [Google Scholar]
  • 22.Walling LL. 2008. Avoiding effective defenses: strategies employed by phloem-feeding insects. Plant Physiol. 146, 859–866. ( 10.1104/pp.107.113142) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Mayer RT, Inbar M, McKenzie CL, Shatters R, Borowicz V, Albrecht U, Powell CA, Doostdar H. 2002. Multitrophic interactions of the silverleaf whitefly, host plants, competing herbivores, and phytopathogens. Arch. Insect Biochem. Physiol. 51, 151–169. ( 10.1002/arch.10065) [DOI] [PubMed] [Google Scholar]
  • 24.Kanno H, Satoh M, Kimura T, Fujita Y. 2005. Some aspects of induced resistance to rice blast fungus, Magnaporthe grisea, in rice plant infested by white-backed planthopper, Sogatella furcifera. Appl. Entomol. Zool. 40, 91–97. ( 10.1303/aez.2005.91) [DOI] [Google Scholar]
  • 25.Chen F, D'Auria JC, Tholl D, Ross JR, Gershenzon J, Noel JP, Pichersky E. 2003. An Arabidopsis thaliana gene for methylsalicylate biosynthesis, identified by a biochemical genomics approach, has a role in defense. Plant J. 36, 577–588. ( 10.1046/j.1365-313X.2003.01902.x) [DOI] [PubMed] [Google Scholar]
  • 26.Park SW, Kaimoyo E, Kumar D, Mosher S, Klessig DF. 2007. Methyl salicylate is a critical mobile signal for plant systemic acquired resistance. Science 318, 113–116. ( 10.1126/science.1147113) [DOI] [PubMed] [Google Scholar]
  • 27.Forouhar F, et al. 2005. Structural and biochemical studies identify tobacco SABP2 as a methyl salicylate esterase and implicate it in plant innate immunity. Proc. Natl Acad. Sci. USA 102, 1773–1778. ( 10.1073/pnas.0409227102) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Mutti NS, et al. 2008. A protein from the salivary glands of the pea aphid, Acyrthosiphon pisum, is essential in feeding on a host plant. Proc. Natl Acad. Sci. USA 105, 9965–9969. ( 10.1073/pnas.0708958105) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Atamian HS, Chaudhary R, Dal Cin V, Bao E, Girke T, Kaloshian I. 2013. In planta expression or delivery of potato aphid Macrosiphum euphorbiae effectors Me10 and Me23 enhances aphid fecundity. Mol. Plant Microbe Interact. 26, 67–74. ( 10.1094/Mpmi-06-12-0144-Fi) [DOI] [PubMed] [Google Scholar]
  • 30.Elzinga DA, De Vos M, Jander G.. 2014. Suppression of plant defenses by a Myzus persicae (green peach aphid) salivary effector protein. Mol. Plant Microbe Interact. 27, 747–756. ( 10.1094/Mpmi-01-14-0018-R) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Rodriguez PA, Stam R, Warbroek T, Bos JIB. 2014. Mp10 and Mp42 from the aphid species Myzus persicae trigger plant defenses in Nicotiana benthamiana through different activities. Mol. Plant Microbe Interact. 27, 30–39. ( 10.1094/Mpmi-05-13-0156-R) [DOI] [PubMed] [Google Scholar]
  • 32.Bos JIB, Prince D, Pitino M, Maffei ME, Win J, Hogenhout SA. 2010. A functional genomics approach identifies candidate effectors from the aphid species Myzus persicae (green peach aphid). PLoS Genet. 6, e1001216 ( 10.1371/journal.pgen.1001216) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Azevedo C, Betsuyaku S, Peart J, Takahashi A, Noël L, Sadanandom A, Casais C, Parker J, Shirasu K. 2014. Role of SGT1 in resistance protein accumulation in plant immunity. EMBO J. 25, 2007–2016. ( 10.1038/sj.emboj.7601084) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Pietrzykowska M, Suorsa M, Semchonok DA, Tikkanen M, Boekema EJ, Aro EM, Jansson S. 2014. The light-harvesting chlorophyll a/b binding proteins Lhcb1 and Lhcb2 play complementary roles during state transitions in Arabidopsis. Plant Cell 26, 3646 ( 10.1105/tpc.114.127373) [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material
rstb20180314supp1.pdf (787.8KB, pdf)

Data Availability Statement

The data cited in this research are available from the Short Read Archive of NCBI accession no. SRP149658.

Articles from Philosophical Transactions of the Royal Society B: Biological Sciences are provided here courtesy of The Royal Society

RESOURCES